The invention relates to the discovery that certain biological toxins specifically bind to the glycosylphosphatidylinositol (GPI) anchor component of certain cell-surface proteins. Applications of this discovery include the detection and diagnosis of paroxysmal nocturnal hemoglobinuria.
Aerolysin is a channel-forming cytolytic protein produced by virulent Aeromonas species, such as Aeromonas hydrophila. Aerolysin is one of the best studied of all of the bacterial cytolytic toxins. It is known to be secreted as a 52 kDa precursor called proaerolysin; this precursor form is converted to the active form by proteolytic removal of a C-terminal peptide. Many eucaryotic proteases can activate proaerolysin, as can proteases secreted by A. hydrophila itself. Once bound to a susceptible cell, aerolysin is transformed into an insertion-competent state by oligomerization. The oligomers, which are heptameric, bridge the lipid bilayer, producing discrete 1 nm channels which result in cell lysis.
It was previously believed that aerolysin bound specifically to certain proteins found on the cell surface, such as the Thy-1 antigen (see U.S. Pat. No. 5,798,218). The present invention, however, is founded on the discovery that the target for aerolysin binding is not any particular cell surface protein, but is actually the glycosylphosphatidylinositol (GPI) anchor that is a component of many cell surface proteins. As their name suggests, GPI anchors function to anchor proteins into the cell membrane; typically the GPI anchor component is at least partly embedded in the membrane, permitting the extracellular component of the protein to be presented to the surrounding environment.
Since GPI anchors are components of many important cell surface proteins, this discovery permits the detection of such proteins (or the determination of their absence) through the use of specific binding assays. For example, since aerolysin typically lyses cells following binding to the GPI anchored protein, one such assay is based on the differential rates of lysis observed when aerolysin is mixed with cells that either have, or do not have GPI anchored surface proteins. While such assays may be performed using aerolysin, other toxins that are related to aerolysin may also be employed for such methods. Such toxins include Clostridium septicum alpha toxin (Parker et al., 1996), and enterlobin (a cytolytic protein produced by the Brazilian tree Enterolobium, Sousa et al., 1994). The binding specificity of these toxins and the use of this specificity to detect GPI anchored proteins is of particular clinical relevance for the disease paroxysmal nocturnal hemoglobinuria (PNH).
PNH is an acquired hematopoietic stem cell disorder manifested by abnormal hematopoiesis, complement-mediated intravascular hemolysis and a propensity toward thrombosis (Rosse, 1997). The disease usually results from a somatic mutation in the X-linked gene, PIGA (Miyata et al, 1993; Takeda et al., 1993; Miyata et al., 1994;
Bessler et al., 1994) The product of the PIGA gene is necessary for the first step in the biosynthesis of GPI anchors. Hence, cells harboring PIGA mutations are characterized by a deficiency, absolute or partial, of all proteins affixed to the cell membrane by GPI anchors. The cells affected by this mutation include erythrocytes, granulocytes, monocytes and lymphocytes. PNH is closely associated with a range of hematological disorders, including aplastic anemia, certain leukemias and myelodysplasic syndrome, and assays for the disease are routinely performed for patients manifesting hematological disorders.
GPI anchored proteins have been shown to be involved in a wide range of important cell functions, including signal transduction (Robinson, 1991; Stefanova et al., 1991 ), and trafficking of apically expressed epithelial proteins (Brown et al., 1989; Powell et al., 1991). They may also play a role in regulating apoptosis (Brodsky, 1997). Two GPI anchored proteins, CD55 (decay accelerating factor) and CD59 (membrane inhibitor of reactive lysis), normally protect cells from the action of homologous complement, and it is their absence that leads to the hemolytic anemia associated with PNH (Rosse, 1982; Rosse, 1995).
The initial mutation of the PIGA gene occurs in a pluripotent hematopoietic stem cell. This cell subsequently divides and gives rise to multiple hematopoietic lineages. These various lineages generate lymphocytes, such as B cells and T cells, leukocytes and erythrocytes, all of which may be GPI anchor deficient (Rosse, 1997). Therefore, in a blood sample from a PNH patient there will be both GPI anchor deficient lymphocytes (those derived from the affected stem cell), as well as normal lymphocytes (those derived from an unaffected stem cell). Furthermore, cells derived from the affected stem cell generally fall into one of three categories. These categories are defined by the cells sensitivity to complement (I=normally sensitive cells, II=cells of intermediate sensitivity, and III=very sensitive cells).
Another characteristic of PNH is a decrease in erythrocyte survival (Rosse, 1971), and a normal or even increased survival of granulocytes (Brodsky, 1997: Horikawa et al., 1997; Brubaker et al., 1977). This decrease in erythrocyte survival poses a problem to clinicians because the two most popular methods of diagnosing PNH, the Ham""s test and the sucrose hemolysis test, involve isolating erythrocytes and measuring their sensitivity to homologous complement. Because of decreased erythrocyte survival in PNH patients, the results from these assays do not provide accurate information about the percentage of affected blood cells. Furthermore, these assays are relatively insensitive and may not detect small populations of PNH cells, such as may be present at the early stages of the disease.
In addition to the Ham""s test and the sucrose hemolysis test, flow cytometry is sometimes used to diagnose PNH. Flow cytometry offers the clinician the ability to use monoclonal antibodies to a variety of different GPI anchored proteins. These antibodies can be used to detect GPI anchored proteins on a variety of different cell types. For example, monoclonal antibodies to CD59 can be used to detect GPI anchors on granulocytes and other cells, thus providing a more accurate assessment of the number of PNH affected cells. Unfortunately, however, flow cytometry requires expensive equipment and significant technical expertise that is not available in many laboratories. Additionally, while flow cytometry can be significantly more sensitive than the sucrose hemolysis and Ham""s tests, it cannot be routinely used to detect PNH cells in populations of less than 1-2% of total cells (Schubert et al., 1991; Hall et al., 1996). Therefore, there is a need for an assay that is inexpensive, accurate and specific for the detection of small populations of PNH affected cells, and the present invention provides such an assay.
The invention provides a number of ways of detecting the presence of PNH cells in biological samples. Generally, these methods comprise contacting a biological sample containing blood cells with a toxin that specifically binds to GPI anchored proteins, and monitoring binding of the toxin to the blood cells. In view of the specificity of the toxin, decreased binding of the toxin to the blood cells compared to binding observed with a control blood sample indicates decreased GPI anchored proteins, and thus the presence of PNH cells.
The biological sample will typically be taken from an individual who is to be screened for PNH, and may be, for example, whole blood, granulocytes or erythrocytes. The toxin may be aerolysin, proaerolysin, Clostridium alpha toxin, enterolobin, or any other toxin that specifically binds to GPI anchored proteins. While the toxin will generally be in its naturally occurring form, forms of the toxin having an altered amino acid sequence may also be employed. For example, forms of aerolysin that bind to GPI anchored proteins, but which do not lyse cells to which they bind are known, and may be used in the assay.
Where a normal (cytolytic) form of the toxin is employed, monitoring the binding of the toxin to the blood cells may be achieved by monitoring the amount or rate of lysis of the blood cells. Typically, the blood cells in a sample taken from a healthy patient will lyse in the presence of aerolysin, since the toxin will bind to GPI anchored proteins present on the surface of such cells, and subsequently insert into the membrane, forming holes that result in cells lysis. PNH cells, on the other hand, will be resistant to lysis because of the deficit of GPI anchored proteins compared to the cells from a healthy patient. Toxin-induced lysis results in a clearing of the test fluid (decrease in optical density), and may be readily monitored by standard means, including visual inspection, microtiter plate readers or spectrophotometers.
By way of example, one embodiment of the present invention comprises an assay for PNH cells in which a range of dilutions of a suitable toxin (such as aerolysin) is utilized. Such a dilution range may be conveniently established in a microtiter plate. The blood sample to be tested is then added to the various dilutions and the mixtures are incubated. Typically, a parallel control experiment is performed in which a blood sample from a healthy individual is utilized. Following incubation, the sample and controls are compared for lysis, and the lowest concentration of toxin at which lysis is observed is noted. If the sample is observed to lyse at higher concentrations of toxin than the control (or to be resistant to lysis), then the patient from whom the sample was taken may be considered to be at risk of having PNH, and further investigation is warranted.
Representative ranges of toxin concentrations that can be used in such an assay are for example, 1 M toxin to 1xc3x9710xe2x88x9211 M toxin, 1xc3x9710xe2x88x924 M toxin to 1xc3x9710xe2x88x9211 M toxin, and 1xc3x9710xe2x88x926 M toxin to 1xc3x9710xe2x88x9210 M toxin. The number of different dilutions used can vary depending on the degree of precision desired. In other words very small increments between two sequential dilutions can be used to provide more precise results. Conversely, a rough estimate of toxin resistance can be determined using as few as two different concentrations of toxin.
In another embodiment of the invention, a sample can be treated with a toxin specific for GPI anchored proteins and the rate of lysis can be determined by measuring optical density at various time points. A sample of cells having a high concentration of GPI anchored proteins will lyse more quickly than a sample of cells having a low concentration of GPI anchored proteins. Therefore, comparing the rate of lysis observed with the rate observed with a control permits the relative concentration of GPI anchors to be determined.
Alternatively, the binding of a toxin specific to GPI anchored proteins may be detected and/or quantified by employing a form of the toxin conjugated to a detectable label (such as a fluorescent or radioactive label). Detection of the cell-toxin-label complex may then be accomplished by any standard means, including flow cytometry and fluorescence activated cell sorting (FACS). Such assays are typically best performed where lysis of the cells is avoided; this may be achieved by using a non-cytolytic form of the toxin (such as the precursor form, or a mutant), or by performing the assay at a reduced temperature (e.g., 4xc2x0 C. or less) at which the toxin will bind to GPI anchored proteins, but will not cause cell lysis.
In a further embodiment of the invention, a cytolytic toxin that specifically binds to GPI anchored proteins, such as aerolysin, can be used to enhance the sensitivity of other PNH assays, such as flow cytometry as described by Hall and Rosse (1996). Since aerolysin will preferentially lyse normal (i.e., non PNH) cells in the sample, pre-incubation of the sample with aerolysin will increase the relative concentration of PNH cells, making such cells more easily detected by the assay.
In addition to methods for detecting PNH cells, the present invention provides methods for generally detecting or quantifying the presence of GPI anchored proteins in a biological sample. Such methods typically comprise contacting the biological sample with a toxin that specifically binds to GPI anchor-containing proteins and detecting or quantifying the binding of the toxin to GPI anchored proteins. Detecting or quantifying the binding of the toxin to GPI anchored proteins may be accomplished by methods including detecting lysis of cells (if a cytolytic form of toxin is employed), or detecting the presence of a detectable label in a GPI anchor/toxin/label complex.
The methods provided by the invention may further be used to distinguish between cells having GPI anchored proteins (GPI+ cells) and cells lacking GPI anchored proteins (GPIxe2x88x92 cells), and may be applied to separate and sort such cells. By way of example, a cell mixture may be incubated with a non-cytolytic form of aerolysin conjugated to a fluorescent marker. Following binding of the aerolysin to GPI anchored proteins, the cells may be separated into collections of GPI+ and GPIxe2x88x92 cells using conventional flow cytometry methods.
In another embodiment of the invention, a cytolytic toxin that specifically binds to GPI anchored proteins, such as aerolysin, can be used to detect small populations of cells that are GPI anchor deficient. Finding cells that are GPI anchor deficient can be indicative of a genetic mutation that affects the presentation of GPI anchors on the cell surface. Hence, this particular embodiment is useful for determining genetic variations that affect the expression of GPI anchored proteins prior to an actual physical manifestation of disease. Used in this way, the invention involves pre-incubating a mixture of cells with a toxin specific for GPI anchored proteins. This pre-incubation, results in lysis of the cells that express GPI anchored proteins, and effectively increases the proportion of GPI anchor deficient cells to a level that can be detected using various other means.
These and other aspects of the invention are illustrated by the following examples and descriptions.